Asc1p/RACK1 Connects Ribosomes to Eukaryotic Phosphosignaling

Area of Science:

• Molecular Biology
• Cellular Biology
• Biochemistry

Background:

• WD40 repeat proteins form beta-propeller structures involved in eukaryotic cellular adaptation.
• RACK1 protein (Asc1p in Saccharomyces cerevisiae) is a seven-bladed beta-propeller located at the ribosomal 40S subunit head.
• Asc1p plays a role in regulating cellular signaling pathways.

Purpose of the Study:

• To investigate the role of Asc1p phosphorylation in its stability and function.
• To identify the impact of Asc1p phosphorylation on other cellular proteins.
• To elucidate Asc1p's function as a control point in ribosomal phosphosignaling.

Main Methods:

• Site-directed mutagenesis to create ribosomal binding-compromised variants (Asc1DEp).
• Analysis of protein stability and degradation pathways (e.g., proteasomal degradation).
• Phosphoproteomic analysis to identify Asc1p-influenced phosphorylation sites on other proteins.

Main Results:

• Mutation of phosphosite T143 to alanine destabilized Asc1DEp, likely via proteasomal degradation, causing asc1- phenotypes.
• Phosphosite Y250 influenced resistance to translational inhibitors but not Asc1DEp stability.
• Asc1p phosphorylation status influenced the phosphorylation of approximately 90 proteins at 120 sites, affecting key cellular processes.

Conclusions:

• Asc1p/RACK1 is a critical phosphosignaling hub regulated by post-translational modifications.
• Asc1p acts as a control point on the ribosomal 40S subunit, integrating signals for cellular adaptation.
• Asc1p's phosphorylation regulates diverse cellular functions including translation, protein turnover, and signaling.